The present invention relates to the detection of analytes in biological fluids. More specifically, the invention relates to enzyme substrate delivery and product registration in one step enzyme immunoassays.
Analyte-specific binding assays are important tools for detecting and measuring environmental and biologically relevant compounds, including hormones, metabolites, toxins and pathogen-derived antigens. A convenient version of the binding assay is an immunoassay which can be conducted in a xe2x80x9clateral flowxe2x80x9d format.
Devices useful for performing lateral flow assays typically include several xe2x80x9czonesxe2x80x9d that are defined along a length of a matrix. The matrix defines a flow path and provides fluid connection between the various zones, including a sample receiving zone, a labeling zone for specifically labeling the analyte, and a capture (detection) zone located downstream from the sample receiving zone and the labeling zone. An absorbent zone (sink) typically is located downstream of the capture zone, and provides a means for removing excess sample and unbound label from the matrix.
In some applications the matrix of a lateral flow assay device is a membrane capable of xe2x80x9cnon-bibulous lateral flow.xe2x80x9d In these applications liquid flow occurs such that all of the dissolved or dispersed components in the analyte-containing liquid are carried at substantially equal rates and with relatively unimpaired flow laterally through the membrane. This is distinguished from a situation wherein preferential retention of one or more components occurs, for example, in materials capable of adsorbing or imbibing one or more of the components.
A principal advantage of the lateral flow immunoassay is the ease with which the testing procedure is carried out. In this procedure a fluid sample first contacts the matrix following application to the sample receiving zone. Capillary action then draws the liquid sample downstream into a labeling zone that contains a means for indirectly labeling the target analyte. The labeling means generally will be a labeled immunoglobulin, but alternatively may be a non-immunoglobulin labeled compound which specifically binds the target analyte. After flowing through the labeling zone, the sample continues to flow into the capture zone where it contacts an immobilized compound capable of specifically binding the labeled target analyte or the complex formed by the analyte and label. As a specific example, analyte-specific immunoglobulins can be immobilized in the capture zone. Labeled target analytes will bind the immobilized immunoglobulins upon entering the capture zone and will be retained therein. The presence of the labeled analyte in the sample typically will be determined by visual detection of the label within the capture zone. Finally, the procedure is complete when excess sample is taken up by the material of the absorbent zone.
Lateral flow immunoassays typically employ test and procedural control lines in the capture zone. The test line serves to detect an analyte present in a test sample, while the procedural control line conventionally serves to detect a ligand unrelated to the analyte. Rather than being applied in the test sample, the ligand unrelated to the analyte is disposed in the labeling zone of the lateral flow immunoassay device. The test line ordinarily employs specific competitive, sandwich or indirect binding separation principles using a visual label. This requires the use of a labeled detector antibody in the labeling pad of the labeling zone and a capture antibody or ligand immobilized at the capture test line.
The capture zone of lateral flow immunoassay devices may also include a procedure control line useful for indicating that a procedure has been performed. The procedure control line generally is located downstream of the binding compound that is immobilized in the capture zone at the test line where reaction occurs. Retention of label by the procedural control line indicates that liquid sample has flowed through the capture zone and contacted the immobilized target-specific binding substance. The accumulation of visible label may be assessed either visually or by optical detection devices.
Another type of enzyme immunoassay utilizes a flow-through device which is described in U.S. Pat. No. 4,632,901. This device comprises a membrane or filter to which an antibody is bound. An absorbent material in contact with the membrane or filter induces flow therethrough when a fluid sample is added to the membrane or filter. A fluid sample is applied to the membrane and, if the cognate antigen is present, is bound by the antibody. A solution of labeled antibody against the antigen is then added followed by a washing step to remove unbound labeled antibody. The presence of labeled antibody on the membrane after washing indicates the presence of the antigen in the sample being assayed.
In one step enzyme immunoassays (EIAs), whether they be flow-through or lateral flow constructs, there is an inherent limitation to the use of enzyme amplification wherein the enzyme (as either enzyme-antibody conjugate or enzyme-label particulate) must be kept separate from its substrate until separation of bound and free enzyme conjugate or label is complete. The present invention addresses methods for such separation.
One embodiment of the present invention is an enzyme immunoassay device, comprising a sample pad comprising a slow lane and a fast lane separated by a hydrophobic barrier, wherein the slow lane contains an enzyme substrate and the fast lane contains an enzyme-antibody conjugate having affinity for an analyte; a capture zone in fluid communication with the sample pad, the capture zone having a capture antibody incorporated therein having affinity for said analyte; and an absorbent zone in fluid communication with said capture zone. Preferably, the analyte is a hormone, enzyme, lipoprotein, bacterial antigen, viral antigen, immunoglobulin, lymphokine, cytokine, drug or soluble cancer antigen. Advantageously, the sample pad comprises high density polyethylene.
Another embodiment of the present invention is a flow-through lateral flow enzyme immunoassay device, comprising a disk comprising inner and outer hydrophilic zones separated by a hydrophobic barrier, the inner zone containing an enzyme substrate and having a smaller pore size than the outer zone, the outer zone containing an enzyme-antibody conjugate having affinity for an analyte; a contact pad in fluid communication with the molded disk; a capture zone in fluid communication with the contact pad; and an adsorbent zone in fluid communication with the contact pad.
The present invention also provides a lateral flow enzyme immunoassay device, comprising a sample pad; a label pad in fluid communication with the sample pad, the label pad containing an enzyme-antibody conjugate having affinity for an analyte; a capture zone in fluid communication with the label pad, the capture zone containing a capture antibody having affinity for the analyte; and an enzyme substrate at a test line. In one aspect of this preferred embodiment, the substrate is chemically immobilized at the test line. Alternatively, the substrate is immobilized in a mordant under the test line. Still alternatively, the substrate is immobilized in a mordant dispensed within the test line. Preferably, the capture zone further comprises chemical groups incorporated therein, the chemical groups capable of specifically reacting with the product resulting from enzyme action on the substrate. Advantageously, the chemical groups comprise diazotized amines.
Another embodiment of the invention is a lateral flow enzyme immunoassay device, comprising a sample pad; a label pad in fluid communication with the sample pad, the label pad containing a substrate covalently attached to a particle or imbibed within a sac, wherein the substrate-containing sac or particle is attached to an antibody; a capture zone in fluid communication with the label pad, the capture zone containing an enzyme/mediator for releasing the substrate and a capture antibody at a test line; and an absorbent zone in fluid communication with the capture zone. Preferably, the sac comprises a liposome. Alternatively, the sac comprises an erythrocyte ghost. Advantageously, the particle label comprises polyalkylcyanoacrylate polymer monosized colloids. The enzyme/mediator may immobilized in a mordant within or under the test line, or may be attached to the capture antibody.
The present invention also provides an enzyme immunoassay device, comprising a sample pad comprising a first lane containing a first barrier zone and a second lane containing a second barrier zone, wherein the first lane contains an enzyme-antibody conjugate having affinity for an analyte and said second lane contains an enzyme substrate, wherein the first barrier zone dissolves before said second barrier zone; a capture zone in fluid communication with the sample pad, the capture zone containing a capture antibody incorporated therein having affinity for the analyte; and an absorbent zone in fluid communication with the capture zone. The barrier zones may comprise structural hydrogel, enterosoluble coatings or biodegradable phospholipids.
Still another embodiment of the invention is an enzyme immunoassay device, comprising a sample pad containing a first enzyme and a second enzyme, the second enzyme conjugated to a second antibody having affinity for an analyte; a label pad in fluid communication with the sample pad, the label pad containing a substrate for the first enzyme, wherein the substrate for the first enzyme is converted by the first enzyme to a second substrate for the second enzyme; and a capture zone in fluid communication with the label pad, the capture zone containing a first antibody having affinity for the analyte at a test line, wherein the second substrate is converted by the second antibody to an enzyme product; and an absorbent zone in fluid communication with the capture zone. Preferably, the first enzyme is alkaline phosphatase, esterase, protease, sulfatase, chymotrypsin-like protease, creatine amidinohydrolase or arginase. Advantageously, the second enzyme is xcex2-D-galactosidase, N-acetylglucosaminidase, xcex1-L-arabinofuranosidase, exglucanase, chitobiosidase, xcex1-L-fucosidase, xcex2-D-glycosidase, xcex1-galactosidase, xcex2-glucosidase, glucansucrase, xcex2-D-glucuronidase, xcex1-amylase, xcex1-mannosidase or xcex2-mannosidase. According to another aspect of this preferred embodiment, the analyte is a hormone, enzyme, lipoprotein, bacterial antigen, viral antigen, immunoglobulin, lymphokine, cytokine, drug or soluble cancer antigen.
The present invention also provides a sample receiving layer for use in an enzyme immunoassay device, comprising: a disk comprising inner and outer hydrophilic zones separated by a hydrophobic barrier, the inner zone containing an enzyme substrate and having a smaller pore size than the outer zone, the outer zone containing an enzyme-antibody conjugate having affinity for an analyte.